McAshan S K, Vergin K L, Giovannoni S J, Thaler D S
Laboratory of Microbiology, Rockefeller University, New York, NY 10021, USA.
Microb Drug Resist. 1999 Summer;5(2):101-12. doi: 10.1089/mdr.1999.5.101.
Handwerger and colleagues demonstrated that a particular clinical isolate of Enterococcus faecium, designated GUC, and here redesignated as GUCR, can conjugatively transfer vancomycin resistance. The vancomycin resistance is encoded by a chromosomally born linked set of genes in the donor, designated the vanA cluster, to the chromosome of an E. faecalis recipient, JH2-2. Here it is reported that an earlier isolate of E. faecium from the same patient who later harbored the vancomycin-resistant E. faecium GUCR lacks the vanA gene cluster but is otherwise similar (by SmaI chromosomal fingerprint and metabolic fingerprinting) to the vancomycin-resistant GUCR. Therefore, "GUCS" is a strong suspect as the base strain for the clinical acquisition of the vanA cluster present in GUCR. Thirteen laboratory-generated vanA transconjugants derived from conjugation between GUCR and JH2-2 were subjected to further analysis, allowing a comparison between transfer in the laboratory and transfer that occurred in the clinical setting. Surprisingly, each JH2-2 transconjugant had a unique constellation of abilities to oxidize various members of a panel of potential carbon sources. This pattern was stable for each transconjugant, and it was not changed by growing the strains with or without vancomycin. Transconjugants had pulsed-field gel electrophoretic (PFGE) patterns largely consistent with that of JH2-2, the recipient in conjugation experiments. However, PFGE analysis showed that a large but variable amount of DNA, between 145 kb and 277 kb, was transferred into different transconjugants. The mechanism appears to be conjugative transposition in which new DNA is added to the pre-existing genome rather than substituting for a segment in the recipient. Mapping and hybridization studies of several transconjugants showed that each received similar, but not exactly the same, DNA fragment of at least 30 kb from the donor. Sequencing of 16S ribosomal genes was used to confirm that the recipient and donor strains used in transconjugation experiments were different species. Sequence analysis was also used to consider the possibility that rRNA operons might be mobilized in conjugation, but no evidence for the transfer of rDNA operons was found. An apparent insertion sequence in E. faecium almost identical to IS 1485 and 57% sequence identity to IS 199 of Streptococcus mutans was found in the region of DNA transferred. The results imply new consequences of conjugative transfer and interspecies recombination.
汉德韦格及其同事证明，粪肠球菌的一种特定临床分离株，最初命名为GUC，现重新命名为GUCR，能够通过接合作用传递万古霉素耐药性。供体中万古霉素耐药性由一组位于染色体上的连锁基因编码，称为vanA簇，该基因簇可转移至粪肠球菌受体JH2 - 2的染色体上。据报道，同一患者先前分离出的一株粪肠球菌，该患者后来携带了耐万古霉素的粪肠球菌GUCR，该菌株缺乏vanA基因簇，但在其他方面（通过SmaI染色体指纹图谱和代谢指纹图谱）与耐万古霉素的GUCR相似。因此，“GUCS”极有可能是GUCR中存在的vanA簇临床获得的基础菌株。对13株由GUCR与JH2 - 2接合产生的实验室衍生vanA转接合子进行了进一步分析，从而能够比较实验室中的转移情况与临床环境中发生的转移情况。令人惊讶的是，每个JH2 - 2转接合子氧化一组潜在碳源中各种成员的能力组合都独一无二。这种模式对每个转接合子都是稳定的，并且在有或没有万古霉素的情况下培养菌株时都不会改变。转接合子的脉冲场凝胶电泳（PFGE）图谱与接合实验中的受体JH2 - 2基本一致。然而，PFGE分析表明，大量但可变的DNA（145 kb至277 kb之间）被转移到了不同的转接合子中。其机制似乎是接合转座，即新的DNA添加到预先存在的基因组中，而不是替代受体中的一段DNA。对几个转接合子的图谱绘制和杂交研究表明，每个转接合子都从供体接收了至少30 kb的相似但不完全相同的DNA片段。对16S核糖体基因进行测序以确认转接合实验中使用的受体和供体菌株是不同的物种。序列分析还用于考虑rRNA操纵子在接合过程中可能被转移的可能性，但未发现rDNA操纵子转移的证据。在转移的DNA区域发现了粪肠球菌中一个明显的插入序列，它与IS 1485几乎相同，与变形链球菌的IS 199有57%的序列同一性。这些结果暗示了接合转移和种间重组的新后果。
